Difference in Primer Melting Temperatures - Maximum allowable Tm difference? (Feb/26/2009 )

Hello,

What is the maximum allowable melting temperature difference between forward and reverse primers?

I've obtained a pair of degenerate primers with a 4 degree difference in melting temperature (e.g. 55 vs 59). (Temperatures were calculated by the manufacturer).

Could this be a problem?

I'm planning on running a temperature gradient to determine the best annealing temperature; can anyone please suggest what annealing temperatures should I try?

Many thanks!

rustyshackleford on Feb 26 2009, 02:57 PM said:

I do not know the maximum allowable difference. But the maximum difference that I would allow is 2 Celsius. In situations where there is a difference in primer tm, use the lower tm. I would try using a melting temperature of 52 C.

As for a temperature range
50C, 52C, 54C, 56C,

Could this be a problem, yes it can. Will it be a problem... well you will have to give it try and find out.

Hey

Hey

I have gone upto a difference of 4 degree C and generally use 4 degrees less than the lower Tm for annealing.

TC

perneseblue on Feb 27 2009, 06:40 AM said:

rustyshackleford on Feb 26 2009, 02:57 PM said:

It should be ok. I had used a short forward primer(Tm 50) and a long reverse primer (Tm 65) because I want to add some extra sequence at the 3' end. The annealing temp was 55, and the amplification was good.

Hi all,

Thank you for your replies and advice!

I'll will definitely just have to give it a try and find out!

Cheers!